EXPERtMENTAL Leprosmaom.
5153105231“ gANtCOLA

INFECTION IN CALVES

Thcsts for “10 Davao of M. .5.
MICHIGAN STATE UNIVERSITY
Salad"; Eidin Embabi
1964

- .
I --.‘.‘-_-

THESIS

 

LIBRARY

Michigan State
University

 

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ABSTRACT

EXPERIMENTAL LEPTOSPIROSIS:
LEPTOSPIRA CANICOLA INFECTION
IN CALVES

 

by Salah Eldin Imbabi

A study of experimental L, canicola infection was
conducted on ten calves. Seven of these were infected by
the subcutaneous route using inoculums of leptOSpiremic
hamster blood. Clinical, bacteriologic, hemotologic,
serologic and pathologic aSpects of the disease were
observed.

Clinical symptoms were manifested 14 to 24 hours
following inoculation, and lasted up to postinoculation
(P.I.) day five or six. Marked thermal response, temporary
anorexia, general weakness, lethargy, stiffness of legs
and in two calves, diarrhaea, were observed.

LeptOSpiremia was detected in all infected calves
12-24 hours after inoculation and it continued up to P. I.
days four or five. LeptOSpiruria started in all infected
calves between P. I. days 12 and 20 and was detectable up
to day 37 in some calves. LeptOSpires were also in the
brain of one calf on day six and in the kidney up to day
40 but the liver and spleen were negative as early as P. I.

day six, as shown by guinea pig inoculation.

Salah Eldin Imbabi

Specific serum antibodies against E. canicola were
observed in significant titers consistently on the sixth
day after inoculation in all infected calves. Maximum
titers of up to 106 were recorded;

Moderate anemia with varying reductions in packed
cell volume, hemoglobin and erythrocyte counts, was seen
but there was no hemoglobinuria or jaundice. There was an
initial leukocytosis with marked neutrophilia that lasted
for two or three days followed by leukopenia which continued
up to around day 10. A slight absolute lymphopenia and
monocytosis were observed.

No impairment to renal function was noticed. This
was evaluated by measurements of blood urea nitrogen and
examination of urine for albumin, Specific gravity, and pH,
all of which gave normal results. Hepatic function was
tested by the bromosulphalein clearance technique.
Variable increases in the half time values, were observed
in some instances, that did not correlate with other
findings. The value of the data obtained on liver function
in this experiment may be questionable.

At necrOpsy no significant lesions were found except
in the kidney where small greyish foci were seen in the

cortex. These foci extended into the medulla.

EXPERIMENTAL LEPTOSPIROSIS:

LEPTOSPIRA CANICOLA INFECTION

 

IN CALVES

By

Salah Eldin Imbabi

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Surgery and Medicine

196A

\
V ‘f _
L) (In.

ACKNOWLEDGMENTS

The author wishes to express his graditude to Dr.

G. H. Conner, Department of Surgery and Medicine for his
generous help, guidance, and continued encouragement.

The author is also indebted to Dr. S. D. Sleight,
Department of Veterinary Pathology for his help, advice,
constructive criticism; for his aid in the histopathologic
examination of tissues, and for furnishing the photomicro—
graphs.

The valuable help and encouragement, extended by
Dr. D. A. Schmidt, Department of Veterinary Pathology and
his assistance with the B. S. P. Determinations is highly
appreciated.

Many thanks are due to Mrs. A. M. Lundberg, Depart-
ment of Microbiology for her technical assistance and to
Mrs. M. J. Long, Department of Veterinary Pathology for

her help with the determinations of blood urea nitrogen.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS.

LIST OF TABLES

LIST OF FIGURES.

LIST OF APPENDICES.

Section
INTRODUCTION.
LITERATURE REVIEW
MATERIALS AND METHODS.
EXPERIMENTAL RESULTS
DISCUSSION
SUMMARY AND CONCLUSIONS

BIBLIOGRAPHY.

APPENDICES

iii

Page
ii

iv

vii

16
21
29
39
65
74

Table

10.

LIST OF TABLES

Summary of Leptospiremia,Leptospiruria,
and Serum Antibody in Infected Calves

Summary of Reductions in Hematological
Values of Experimental Calves.

Postinoculation Days on Which Occult Blood
was Demonstrated in Urine of Infected
Calves

Mean (X) and Standard Deviation (O) of the
Absolute Leukocyte Counts and Hematologic
Values of Control Calves . .

Mean (X) and Standard Deviation (O) of the
Absolute Differential Leukocyte Counts

and Hematologic Values of Infected Calves.

Summary of Renal Function Test; Mean Values
of B.U.N. Before and After Infection, in
mg/lOO ml. of Blood . . . .

Summary of Liver Function Test; Mean, Pre-
and Postinoculation Half Times for
Clearance of Bromsulphalein in Minutes.

Duration of LeptOSpiruria in Experimental
Calves Confirmed by Guinea Pig
Inoculations

Antibody Titers for L. Canicola in Sera of
Infected Calves . . . . . .

Summary of Persistence of L. Canciola in
Tissues of Infected Calves.

iv

Page

41

42

43

44

45

46

47

48

49

Figure

10.
ll.

12.

13.

LIST OF FIGURES

Mean Values of Daily Temperatures of
Experimental Calves

Mean Hemoglobin Levels of Experimental
Calves, Expressed as Percent of Pre—
inoculation Values

Packed Cell Volume; Daily Mean Values,
Expressed as Percent of Pre—inoculation
Levels

Mean Values of Daily Erycthrocyte Counts
Expressed as Percent of Pre—inoculation
Levels

Mean Values of Daily Leukocyte Counts of
Experimental Calves, Expressed as
Percentage of Pre—inoculation Levels

Daily Mean Values for Total Neutrophils
Expressed as Percentages of Pre-
inoculation Values

Daily Mean Values of Lymphocyte Counts
Expressed as Percentages of Pre-
inoculation Values

Showing Daily Mean Values of Monocytes
Expressed as Percent of Pre—inoculation
Counts

Eosinophils;Mean Values of Daily Counts,
Expressed as Percentages of Pre—
inoculation Levels

Kidney Section, Calf 921

Kidney Section, Calf 921

Testicle, Calf 921 . . . . . .

Kidney Section, Calf 922 .

V

Page

56

57

6O
61
61
62
62

Figure
14.

15.
16.

17.

Page
Liver Section, Calf 922 . . . . . . . 63
Liver Section, Calf 922 . . . . . . . 63
Medulla of Adrenal Gland, Calf 922 . . . 64
Kidney Section, Calf 923. . . . . . . 64

vi

Appendix
1.

IO.

ll.

l2.

13.

LIST OF APPENDICES

Daily Temperatures of the Experimental
Calves

Daily Hemoglobin Values of Experimental
Calves

Daily Packed Cell Volumes (P.C.V.) of
Experimental Calves. . . .

Erythrocyte Counts of Experimental Calves

Daily Total Leukocyte Counts of Experimental
Calves . . . . . . . .

Daily Segmented Neutrophil Counts of
Experimental Calves. . . .

Non—segmented NeutrOphil Counts.
Lymphocyte Counts of Experimental Calves.
Daily Monocyte Counts of Experimental Calves

Daily EosinOphil Counts of Experimental
Calves . . . . . . . .

Results of Urin Examination Showing pH and
Specific Gravities . . . .

Renal Function Test; Levels of Blood Uria
Nitrogen (B.U.N.) of Experimental Calves.

Results of Liver Function Test Showing Half

Time Values for Bromosulphalein
Clearance

vii

Page

7C»

76

77
78

79

8o
81
82
83

84

85

86

87

INTRODUCTION

Outbreaks of leptOSpirosis in cattle, where L.
canicola was shown to be the only cause of the disease,
have been reported. The infection was confirmed both
serologically and by actual isolation of organisms from
sick calves.

Experimental evidence, however, was very scanty.
Reports of only three calves infected on two different
occasions were encountered. Nowhere in the literature was
there any report of a controlled experiment to study the
various clinical, clinical pathologic or immunological
aspects of L. canicola infection in cattle.

The experimental work undertaken by the author is

an attempt to meet this deficiency.

LITERATURE REVIEW

The leptOSpiroses are a group of diseases, caused by
a variety of leptOSpiral serotypes.

The leptOSpiral etiology of Weil's disease was first
recognized by Inada, t l. (1916), who classified the

organism as Spirochaeta icterohaemorrhagiae. Since that

 

time many serotypes of leptOSpira were recognized in man
and other animals in many parts of the world. Mitchen and
Azinov (1935) in Russia, were the first to associate lepto-
Spires with icterohemoglobinuria in a calf. Shortly after
-that, Clayton and Derrick (1937), reported the isolation
ofleptOSpira from a farmer near Pomona in Australia and
designated this serotype as L. pomona. Jungherr (1944),
was the first to recognize leptospires in stained kidney
sections from three fatal bovine cases. Baker and Little
(1948) isolated leptOSpires from cows suffering from a
febrile disease characterized by thick blood-stained milk.
This serotype was later identified by Gochenour, _£__l,
(1950), as antigenically similar to ;. pomona.

LeptOSpira are associated with many animal hosts,
as revealed by Steele (1958). The concept that had pre-
vailed in the past concerning host specificity is no longer
generally accepted, Galton, g§_al. (1958). More and more
evidence is accumulating, both of naturally occurring and

2

3
induced infections in host species by various leptOSpiral
serotypes. Faine (1962) considered that the host of election
in leptOSpirosis should be regarded as a result of a quanti—
tative rather than a qualitative adaptation of host and
parasite to one another.

Serologic evidence furnished by Alexander, §£_§l,
(1962), showed the extensive occurrence of L. seJroe agglu-
tinins in bovine sera from ten states. Prior to that, Bryne
and Chambers (1959) had shown the prevalence of this sero-
type in cattle of Maryland, a finding that had been reported
from Florida by Galton, _t_al, (1956); from Georgia by Hale
(1957); and Illinois by Ferris, et_al, (1958). Leptospgra
hardjo was isolated from cattle in Louisiana by Roth and
Galton (1960) and from a cow showing clinical symptoms of
leptOSpirosis on a Pennsylvania farm, Clark, t l. (1961).

Mitchell, t al. (1960), described an outbreak of

leptOSpirosis in cattle in Canada, associated with a sero-

type of the hebdomadis group, namely L. sejroe. Most of

 

the infections, however, were caused bY.E' pomona but they

have also encountered weak serologic reactions against L.

canicola.

LeptOSpira pomona caused widespread epizootics in

 

swine, Bohl and Ferguson (1952) and Yager, _§__1, (1952);
and in cattle, York (1951), Gochenour (1950 and 1953), Rein-
hard (1952 and 1953), and Boulanger (1958). Leptospira

pomona was also found to be the cause of an outbreak in

horses with manifestations of systemic disease, Roberts, e:
'aL. (1952). Some cases of leptospiral meningitis in man

were caused by this serotype, Schaeffer (1951) and Schnurren—
berger (1961).

LeptOSpira pomona was isolated from naturally occur-

 

ring canine cases by Murphy (1957) and by Morter, _L__L.

(1959), and successfully used to establish experimental in-

t a}, (1959). Sheep and goats

fections in dogs by Cholvin,
were also susceptible to experimental infection, Morse, e:
a_l_. (1958).

While the foregoing clearly illustrates the point
that a leptOSpiral serotype can infect more than one host
species and that one host may be infected by more than one
serotype; a further example is furnished by L. canicola. It
was first isolated from a sick dog in the Netherlands by
Klarenbeck (1933) and for many years was considered as con-
fined to dogs and man. This concept, however, was later
proved incorrect.

Olitzki (1951) produced serologic and cultural evi-
dence of L, canicola in cattle, dogs, and pigs. Starr
(1953) investigated a serious outbreak in pigs with parallel
high incidence in dogs and human beings, and significant
serum titers in two out of 21 cows examined. In the same
year Wellington, e£_al, (1953) also encountered a serolog-
ically positive case of L, canicola in a cow with symptoms

of "hematuria."

Van der Hoeden (1955a) reported that prior to 1952
the main etiology of leptOSpirosis in Israel was L, gr p—

potyphosa, but that following 1952, L. canicola started to

 

assume increasing importance in many parts of Israel where
it accounted for several outbreaks among cattle in regions
where its incidence in dogs was comparatively low. There
were indications in one outbreak, that one dog which died
from the disease, had contracted it by licking the boots of
his master who was a herdsman, and of another dog con-
tracting the disease by frequenting pens of infected calves.
Infection in Jackals was also common and many showed high

serum agglutinins against L. canicola which was isolated

 

from the kidneys of two. Again, van der Hoeden (1955) des—
cribed four outbreaks that had occurred previously, namely
in 1949, 1952, 1953, and 1954 respectively. In the first
one, a three weeks old calf died after a short illness with
symptoms of extreme weakness, lack of appetite, bumping
pulsation of the heart and intense jaundice. From the
kidneys of this calf a leptospira was isolated and later

identified as L, canicola. The second outbreak involved

 

mainly calveséflxnfllfive months old, some of which showed
severe symptoms including hemoglobinuria and pyrexia and
half of these were either killed or died from the infection.
The third and fourth outbreaks were noticed among older cows.
Symptoms included hemoglobinuria and premature birth with

high serum agglutinins against L. canicola in both outbreaks.

 

The same author also established infection experi-
mentally in a three and one—half month—old calf by ocular
instillation of L, canicola culture obtained from a dog.

The calf started to have a high temperature on the 9th day
after infection and was found to be serologically positive
against L. canicola on P. I. day 18. At necropsy, the
kidneys had lesions of acute and subacute focal interstitial
nephritis and many leptOSpires were seen in the bladder
urine.

Once more van der Hoeden (1955c) described severe
outbreaks in cattle in Israel due to L, canicola with high
incidence in man and pigs and significant titers in appar-
ently healthy horses, mules and donkeys in areas where
cattle were infected. He postulated that cattle, swine,
dogs and other animals were infected by water and vegetation
contaminated by Jackals and dogs and vice versa.

The incidence of L, canicola in dogs and pigs in
Israel was also reported to be strikingly high, van der
Hoeden (1956). In one case, 14 out of 33 symptomless pigs
examined were serologically positive with titers of from
1:2OO to 1:2000. Many pigs examined in slaughter houses were
shown to have evidence of diffuse and focal interstitial
nephritis with tubular degeneration and some glomerulone—
phritis. As for human infections in Israel, L. canicola was
second to L. grippotyphosa and accounted for 31% of the cases

reported by van der Hoeden (1958). He also reported L,

canicola infection in hedgehogs in which 27 out of 142
examined were serologically positive. The organism was
isolated from 21 of these.

In Hungary, Kiszel, 3: EL. (1957), found strong sero—
logic evidence of L. canicola, among other serotypes, in
dogs, pigs, cattle and horses. Bezdenezhnykh, eL_aL. (1956),
incriminated L, canicola, as the major cause of leptOSpiro-
sis in swine in the Sakhalin islands.

In the United States, Williams, _§__L, (1956), while
studying an outbreak of canicola fever in man, was also able
to isolate L, canicola from dogs and swine in addition to
finding significant serum agglutinins in cattle. In this
instance human beings apparently were infected by using a
small stream near which swine and cattle frequently grazed
and which was also frequented by dogs.

A bacteriologically verified case of L, canicola in
a new born calf was described by Turner, I: _L. (1958). The
calf was presented for treatment with a history of blood in
the urine and intense jaundice. The calf, on examination,
had a very low red blood cell count, leukocytosis, and a
high serum level of combined protein bilirubin with no free
bilirubin. Inoculation of urine from the calf into mature
chinchillas resulted in a disease similar to, but more pro—
nounced than that caused by L, pomona. From the chinchillas

L. canicola was isolated and used to experimentally infect

two calves, readily establishing the infection with

localization in the kidney. The dam of the original calf
and another cow in the herd were serologically positive for
‘L. canicola. There was also a history of one abortion in
the herd and the death of a dog that was in the same area,
from liver disease.

In addition to farm animals and man, various surveys
have shown the existence of various serotypes of leptOSpira
among wild life Galton (1959). In many cases the exact role
of these animals in the epizootiology and epidemiology of
leptOSpirosis is not quite clear. Abdulla, et_al, (1962),
found that 26 out of 75 deer killed in various parts of
Ontario were serologically positive for L, pomona with kidney
lesions suggestive of leptospirosis in most of them. Lgpgg—

spira ppmona was isolated in four instances by inoculating

 

kidney tissue suspensions in liquid media. Trainer, e£_aL,
(1963), also encountered significant serum titers against

.L. pomona and L. autumnalis in a ratio of four to one in

 

white tailed deer in Wisconsin. Jackals and hedgehogs were
considered as reservoirs for L. canicola in the case of the

former and L, canicola and L, grippotyphosa for the latter

 

in Israel and their important role in the epizootiology of
the respective diseases was described by van der Hoeden
(1955c) and 1958).
Lavrova (1962) found serologic and cultural evidence
of leptospiral infections in rodents, namely Microtus arvalis,

Pitymys majori, Apodemus agrarius and Rattus norvegicus and

 

considered them important in the epizootiology of leptospi—
rosis. Parnas, _L_a}, (1961), after a four year survey of
swamp fever in Poland, reported that aquatic, field and swamp

mammals, principally Mus musculus, Microtus arvalis, Arvicola

terrestia and Microtus arviceps were the chief vectors of L.

 

 

 

 

grippotyphosa, L. icterohaemorrhagiae, L. sejroe and L.
anatum.

Forty—four out of 820 mammals (or 5.4%) examined by
McKeever, _L_aL, (1958), were found positive by isolation
from kidney tissue, for various leptOSpiral serotypes. Opos-
sum were positive for L. ballum and members of the L. mgtlgf
hyos serogroup, grey fox for L. ballum, striped skunk for
'L. ballum and L, pomona, wildcat for L. ballum and L. pomona
and raccoon for L, ballum, L. pomona, L. australis, L.

grippotyphosa and L. hebdomadis serogroup. Reilly (1954)

 

 

had also furnished serological evidence that the raccoon
may be a reservoir for L. canicola.

Some workers have suggested arthrOpod vectors as
having a role in the transmission of leptospirosis. Burgdor-
fer (1956) was able to infect the Argasid tick Ornithodoros
turicata by letting it feed on guinea pigs and hamsters
infected with L, pomona. He showed that the organisms per-
sisted in the tissues of the tick for some time. Krepkogor-
skaia, _e_t_ _a_L. (1957), isolated L. grippotyphosa from the
European tick Dermacentor marginatus S, found on cattle in

Russia, where isolated cases of leptOSpirosis were seen.

10

Infection in nature takes place through the mucous
membranes of the mouth, nose, eyes, and abraded skin, Te
Punga and BiShOp (1953), Stoenner (1957), and Steele (1958).
Donatien, eL_aL, (1951), however, reported that they could
infect guinea pigs by feeding emulsified infected liver and
kidney in addition to infection by the intranasal route.

Transmission by coitus or by infected bulls' semen
vuusreported by Sleight, _L_aL, (1961). They suggested that
the semen became contaminated through contact with urine.

In nature, infection usually takes place either by
direct contact with infected urine, voided by infected ani-
t a},

mals during the period of leptOSpiruria, Moore,
(1956), Te Punga, 331. (1953), Steele (1958), Galton, at
31. (1958), Stuart (1956), or by contact with water that

has been contaminated by urine from Shedding animals, Te
Punga, e§_al, (1953), van der Hoeden (1955), and Williams,
_£__L, (1956). GilleSpie, §L_al, (1957), succeeded in
isolating L, pomona from water known to have been contamin-
ated by cattle shedding leptOSpires in their urine.

The seasonal occurrence of outbreaks of leptOSpirosiS
was mentioned in several places in the literature. Mitchell
(1959) stated that the majority of outbreaks in Canada oc—
curred during the period of August to December. According
to van der Hoeden (1958), outbreaks in Israel were reported

mainly during the wet season. Stoenner, e: aL. (1956), pos—

tulated that the confinement of cattle during fall and winter

ll

favored the Spread of the disease.

In the individual animal, the manifestations of lepto-
Spirosis may be very severe, amOunting to a fulminating syn-
drome with high mortality in young subjects, van der Hoeden
(1955), Stoenner, _t___l_. (1956), and Mitchell (1959). The
disease, on the other hand, may be mild or even inapparent,
and can only be detected by serologic methods, Gochenour
(1953), Boulanger, 333;. (1958), and Seibold, 215.9}.- (1961).
Factors like type of host, age and resistance of the individ-
ual animal, Faine (1962), and the pathogenicity and viru-
lence of the infecting strain, Alexander, 23 a}, (1956), are
thought to influence the outcome of the infection.

Body temperatures of 104-107 F., extreme weakness,
depresSion, fast pulse, diarrhea and stiffness of legs were
among the symptoms reported by various wprkers during the
acute febrile phase which usually lasted for one to eight
days, Gochenour (1953), Reinhard (1953), Moore _Lal, (1956),
and Steele (1958).

Hemoglobinuria and jaundice, usually seen in young
animals, were reported to occur in various outbreaks of lep—
toSpirosiS, Reinhard (1951), Moore, g§_al, (1956), van der
Hoeden (1955), Turner, gt aL. (1958), and Boulanger,=_£__L,
(1958). These were also produced experimentally in young
lambs and pregnant ewes by inoculation of leptOSpira free

filtrates containing hemolysins, Bauer, gt a}, (1961), and
Sleight, g£_aL, (1962), reSpectively. Alexander, t al.

12

(1956), discussed differences in hemolysin production among
various serotypes and among various strains within serotypes.
He thought this to be a function of the virulence of various
serotypes and strains which might explain the differences in
the severity of diseases produced by homologous serotype
strains.
Initial leukocytosis which may be followed by leuko-

penia was reported, York, t l. (1951); Roberts, e£_al,

(1952); Turner, e£_aL, (1958); Lindqvist, t l. (1958);

Sleight, £922.1- (1961); and Bertok, _t__1_. (1961).

In cattle, abortion as a sequel to the acute clinical
leptospirosis was reported by Fennestad, e£_al, (1956), who
could produce it experimentally in two out of 21 pregnant
heifers which aborted 23 and 28 days postinoculation,
reSpectively. In many outbreaks of the disease, however,
abortion may be the only symptom and according to many
workers, it was noticed to take place in the last trimester
of pregnancy, Fennestad, _L__l, (1956); Boulanger, _§._L.
(1957); and Mitchell (1959). Retention of the placenta was
reported by Fennestad (1958) and van der Hoeden (1955);
mummified fetuses by Te Punga and Bishop (1953).

An atypical flaccid mastitis with a marked drOp in
milk which becomes thick, yellowish and sometimes blood—
stained is being recognized as a manifestation of lepto—

spirosis in lactating cows and by some workers it is con-

sidered pathognomonic, Gochenour (1953), Mitchell and

13

Boulanger (1959). Schnurrenberger, e£_§L, (1961), investi-
gated outbreaks of leptOSpirosiS among 47 herds and reported
that 30 herds had abortions as the most predominant Sign;
11 herds had flaccid mastitis and Six herds had breeding
difficulties.

It was repeatedly reported that leptospiral meningitis
was a common sequel in man, Saint Martin (1955); van der
Hoeden (1955); Bertok, t a}, (1961). It was observed in

cattle by Hoag, _E._l- (1954).
Conjunctivitis in dogs and cattle and iridocyclitis
in horses as sequels to leptOSpirosis was described by Chol-
vin, _£__L, (1958); Hoag, IE._l- (1957); and Hensser (1952).
Following infection, leptoSpires can be demonstrated
in the peripheral blood during the acute phase of the dis—
ease, Gochenour, _L'aL. (1953), and Moore, £3 a}, (1956).
In experimental infections, leptOSpiremia was demonstrable
up to postinoculation (P. I.) day 10, York (1951); Cholvin,
32.21-(1959)3 Lindqvist, 3: 3L. (1958); and Sleight, gL_aL.
(1961). During leptospiremia, isolation can usually be
made from many organs, including kidney, liver, spleen and
brain. Leptospires, however, tend to disappear from these
organs, except the kidney, by the end of leptoSpiremia,
Gochenour, $12.91.. (1953). Sleight, _L_.l- (1961), could iso—
late L. pomona from the brain up to P. I. day 18. Such

localization in the kidney, with tubular degeneration, inter-

stitial nephritis, glomerular degenerative changes and

l4

lymphocytic infiltration were the most consistent pathologic
findings in the literature (Baker and Little, 1948; Hadlow
and Stoenner, 1955; van der Hoeden, 1955; and Galton, 3: 3L.
1958).

Following localization, infected animals shed lepto—
Spires in the urine for variable periods and other animals
and human beings may be infected by direct contact or in-
directly through contaminated water and vegetation, Te
Punga (1953); van der Hoeden (1955a); and GilleSpie, §§_§L,
(1957). Morse,_LL_aL. (1957), stated that cattle and swine
may shed leptospires for over 90 days.

Impairment of renal function and hepatic function was
described by LOW:._E._I- (1956), Gleiser (1957) in experi-

mental dogs and by Arean (1962) in guinea pigs infected with

.L. icterohemorrhagiae. They were of the Opinion that the

 

renal insufficiency was due primarily to a reduction in the
tubular maximal excretory capacity, a depression of the
glomerular filtration rate and renal plasma flow leading to
nitrogen retention.

The same authors also found that the jaundice that was
seen in the acute phase of the disease correlated nicely
with histOpathological findings of hepatocellular injury
resulting in functional impairment of the liver. Similar

lesions were noticed in Sheep, Bauer, t l. (1961), and in

e
cattle and sheep, Sleight, §L_aL. (1962), by inoculating

leptOSpiral hemolysins. The latter, however, did not find

15

any indications of inflammatory reaction and postulated that
the centrilobular necrosis and disruption of hepatic cords
were attributable to anoxia as a result of hemolytic anemia.

Infection with a leptospira serotype induces production
of antibodies which are detectable in the serum within a few
days after the onset of the infection Te Punga, e_’_g_a_L.(l953),
Boulanger (1958), Mitchell (1959), and Seibold, _E.§l-
(1961). In experimentally infected animals, serum antibodies
were detected as early as postinoculation day four in ewes
with L. pomona, Smith, _L__L, (1960). In cattle with L.
pomona, agglutinins were detectable between seven to ten days
after the start of the febrile response, Moore, _£._L, (1956).
Steele (1958) found them generally six to twelve days after
onset, reaching maximum titers by the third to the fourth
week. Van der Hoeden (1955) reported a titer of l:20,000
against L, canicola in a sick calf on the sixth day of infec-
tion. A horse experimentally infected with L. pomona was
serologically positive on the ninth day after the febrile
response.

Antibodies can also be demonstrated in the urine,
Rudge (1958), which in many instances may interfere with
isolation of leptospires from the urine, Stuart (1956). It

was reported that infection with one serotype did not protect

against another serotype, Menges t l. (1960).

MATERIALS AND METHODS

Ten healthy calves, Six males and four females ranging
in age from five to eight months and in weight from approxi-
mately 250 to 450 pounds were used. The calves were exam—
ined for internal parasites and received apprOpriate anthe-
lminthic treatment. They were serologically tested and
found negative for antibodies against L, ganicola, L, pomona

and L. icterohemorrhagiae. The calves were divided into

 

three groups by random selection. In the first group were
calves 915, 924, and 925; in the second, calves 916, 918,
and 923; and in the third, calves 919, 920, 921, and 922.
Calves 915 from the first, 918 from the second, and 919 from
the third group served as controls.

Pre—infection values for erythrocytes, leukocytes,
differential leukocyte counts, hemoglobin and packed cell
volumes (PCV), were determined at least twice before, and
once on the day of infection prior to inoculation. Renal
and hepatic functions were similarly evaluated.

LeptOSpira canicola, strain MOULTON which was propa—

 

gated in Stuart's medium (Difco) enriched with 10% rabbit
serum, was transferred to young hamsters, by intraperitoneal
inoculation, where it was maintained by regular passage

until the experiment was concluded.

16

l7

Calves were infected by subcutaneous inoculation of
1.5 m1. of hamsters' heart blood, obtained approximately 48
hours after inoculation and collected under general anes-
thesia in heparinized syringes. The first group of calves
received infected blood from the fourth; the second group
from the 16th, and the third group from the 28th hamster
passage respectively. Control calves received 1.5 ml. each
of noninfected, normal hamster's blood.

Following inoculation, infected and control calves
which were properly segregated were closely observed for
any manifestations of disease or any deviation from the
normal. Body temperatures were taken daily for the first
14 days and after that on various days until the animal was
killed, Appendix I.

Blood was collected aseptically from the jugular vein
and cultured daily for the first 14 days by placing two to
three drOpS in ten m1. of Stuart's medium (Difco) enriched
with 10% rabbit serum. This was then incubated at 30 C.
for at least eight weeks and examined weekly for leptoSpireS
by_darktfield microsc0py. Simultaneously, blood from each
infected animal was inoculated into one hamster on postin—
oculation days two to five. The heart blood of these hamsters
was collected approximately 72 hours later, diluted with
saline, centrifuged and the supernatant fluid examined for

leptOSpireS by dark field microsc0py.

18

Smears for daily differential leukocyte counts were
made from fresh blood, promptly dried and stained with
Wright's stain. One hundred cells from each of two Slides
from each animal were differentially counted, using a battle-
ment system, MacGregor eL'aL. (1940), and the average taken.

Blood samples for hemoglobin (HB) and packed cell
volume (PCV) determinations and for erythrocyte and leuko—
cyte counts, were collected in vacuum tubes containing
ethylenediaminetetraacetate (EDTA) as anticoagulant.

Hemoglobin values in.gm/100 m1. of blood were deter-
mined by the cyanmethemoglobin method and the packed cell
volume by the capillary tube method.

Both erythrocytes and leukocytes were counted, using
an electronic cell counter (Coulter), Mattern, §§_al, (1957),
and Grant §t_§;, (1960) adjusted to thresholds 10 and 35
respectively and anaperturecnuwent setting of seven for
both.

Sera were used for blood urea nitrogen (BUN) determin—
ation by means of an autoanalyzer, Skeggs, §£_al, (1957) and
the results calculated as mg/100 ml.

Urine samples were collected, and portions from each
were diluted in physiological saline and two m1. inoculated
intraperitoneally into young guinea pigs weighing approxi—
mately'250 gmh 'The guinea pigs were killed three to four
weeks later and their sera used for detection of Specific

antibodies against L. canicola. Agglutination and/or lysis

19

of 50% of leptospires in the antigen in a serum dilution of
1:100, was considered positive.

Furthermore, the urines were examined directly by dark
field microsc0py for leptospires and examined for occult
blood, albumins, Specific gravity and pH.

All above determinations, along with serological
tests of the calves' sera by the microsc0pic agglutination—
lysis test, Morse, _L__L. (1955), were conducted daily for
the first 14 days postinoculation and thereafter at variable
intervals until the animal was euthanized.

To assess liver function, ten m1. Bromosulphalein*
solution containing 500 mg. phenoltetrabromphthalein-
disodiumsulfonate was injected aseptically into the left
jugular vein while recording the exact time. Approximately
ten ml. of blood from the right jugular vein was collected
after about five minutes and a second sample approximately
five minutes later, accurately recording the time in minutes
and seconds. The sera from the first and second samples
were separated and the amount of dye contained in each was
determined Spectrophotometrically and the values used to
determine the half time clearance.

The calves were killed and subjected to a thorough

post mortem examination. This was done according to the

following schedule:

 

*Hynson, Westcott and Dunning, Inc., Baltimore, Md.

20

Days Postinoculation

 

Group Control calf 915 75
l Infected calf 924 75
Infected calf 925 50

Group Control calf 918 40
2 Infected calf 923 40
Infected calf 916 30

Group Control calf 919 21
3 Infected calf 921 21
Infected calf 920 14
Infected calf 922 6

At necropsy, samples of bladder urine and portions of
liver, Spleen, kidney and brain were aseptically collected.
These tissues were separately emulsified as 10% by volume
in physiological saline and two m1. of each injected intra-
peritoneally into young guinea pigs which were examined
three to four weeks later for Specific serum antibody
reactions. Samples of lung, brain, liver, Spleen, kidney,
skeletal and heart muscles, rib, thyroid and adrenal glands,
lymph node, testicle and seminal vesicle from males, and
ovaries and uterus from females were collected in apprOpri-

ate fixatives for histOpathological examination.

EXPERIMENTAL RESUITS

Following subcutaneous inoculation with infected
hamster blood, all infected calves showed a marked thermal
response starting as early as 14 hours after inoculation
in calves 920 and 921 and within 24 hours in the rest of
the calves. The pyrexia continued up to the fifth or Sixth
day; the highest being on postinoculation (P. I.) days two
and three. Temperatures of the three control calves remained
normal throughout. Figure 1 shows the temperature curves
for the seven infected and three control calves plotted from
the group averages on P. 1. days one to 13. Appendix 1 shows
actual temperatures throughout the experiment.

During the febrile stage, all infected calves were
visibly sick; with a rough coat, dry muzzle, anorexia, sup-
pressed rumination, grinding of teeth, sluggish movements,
and general lethargy. ReSpiration was shallow and acceler-
ated and the pulse was pounding and fast. Calves 920, 921,
922, and 924 had diarrhea and calves 916, 920, 923, and 925
showed marked stiffness of all four legs on postinoculation
days three, four, and five.

After this acute phase had passed, all infected
calves gradually recovered and except for a visible loSS
in condition they all behaved normally after the seventh day.

21

22

LeptOSpiremia, starting as early as 12 hours (calves
920 and 921) and 24 hours after inoculation for the other
five infected calves, was confirmed both by dark field micro—
scopy of blood cultures and by hamster inoculations. Lepto-
spiremia continued up to and including P. I. day four and
in one case (922) on P. I. day five, after which no lepto—

Spires could be demonstrated, Table l.

Hematologic Findings

 

AS early as the second day (calves 916, 920, 921),
or the third to fourth day after inoculation for the rest of
the calves, the hemoglobin, packed cell volume and erythro—
cyte count started to fall steadily, reaching a maximum
reduction around days six to eight. Maximum per cent reduc—
tions in hemoglobin, packed cell volume, and erythrocytes for
infected and control calves, is Shown on Table 2, and is
illustrated graphically, Figures 2, 3, and 4. The actual
values are Shown in Appendices 2, 3, and 4, and the mean and
standard deviations in Tables 4 and 5.

No hemoglobinuria and no icterus were noticed on any
day throughout the experiment, but occult blood was detected
in a few days in the urine of infected calves as summarized

on Table 3.

Leukocytes

 

Five infected calves showed a noticeable increase in

the total leukocyte counts, starting on P. I. day two and

23

amounting to a 60% increase in the case of calf 921, Appendix
5. This leukocytosis continued for about two to three days
and was gradually diminished to a leukOpenia starting on P.I.
day five with lowest values around P. I. day seven, Figure

5, and Tables 4 and 5.

It can be seen from Appendix 6 and Figure 6 that the
initial leukocytosis was due to an absolute neutrOphilia,
which was accompanied by a Slight left shift in calves 916
and 921, Appendix 7 and Tables 4 and 5.

All calves had a decrease in the number of circulating
lymphocytes starting P. I. days two to four and, except in
one calf (924), the lymphOpenia continued up to around
P. I. day 14. In some of the calves the lymphocytes were
reduced to about 30% of their pre-incoulation values, but
the overall maximum reduction as calculated from mean values
for all calves was about 33%, Figure 7 and Tables 4 and 5.
Appendix 8 shows the absolute values of lymphocytes and it
can be seen that while there was an absolute lymphopenia,
some calves had a relative lymphocytosis after P.I. day
five and throughout the period of leukopenia and neutropenia.

Monocytes, on the other hand, Appendix 9, were.shown
to have a definite rise starting around P. I. day four or
five for most of the calves up to around P. I. day six.

The control calves, however, Showed a Similar but less
pronounced monocytosis, Figure 8 and Tables 4 and 5. All

infected calves experienced an eosinopenia starting P. I.

24

day two up to P. I. day 13 when the eosinOphilS started to

rise, Appendix 10 and Figure 9, and Tables 4 and 5.

Examination of the Urine

 

Except for occult blood that was detected on certain
days, Table 3, the urine pH and Specific gravity, did not
Show any more fluctuations than those seen in the same ani—
mals before inoculation or in the control calves. It may
be of interest, however, to note that the lowest values for
specific gravity in all infected calves occurred during P.I.
days nine to eleven, Appendix 11. No albumin was detected

in the urine throughout the experiment.

Renal Function Test

 

Renal function, measured by the blood urea nitrogen
level, before and after the infection did not reveal any im-
pairment. For all intents and purposes, both infected and
control calves fluctuated similarly and remained well within
the normal levels, Appendix 12. Table 6 Shows mean values

for blood urea nitrogen (BUN) before and after inoculation.

Hepatic Function Test

 

Infected calves had variable increases in the half
time values for bromosulphalein clearance ranging from 0.54
to Slightly over 4.00 minutes. Two of three control calves
had increases of 1.32 and 6.8 minutes respectively. With
regard to individual calves, the more noticeable increase

occurred after P. I. day 13; while in the first 12 days

25

after infection, the delay was slight, Appendix 13. Table
7 shows mean values before and after inoculation.

All ten calves excreted varying amounts of bromosul-
phalein in the urine following intravenous injection of the
dye. Urine was collected during the interval between the
first and second samplings of blood or immediately following
the second. The highest amount measured was 7.6 mg. in 62
ml. of urine which comprised about 1.5% of the total amount
injected. This was easily noticed because of the purple
color it gave to the alkaline urine. Its concentration was

measured SpectrOphotometrically.

Leptospiruria

 

No leptospires were detected by dark field microscopy
in any of the urine samples collected from the calves.
Twenty-one of these samples, however, induced serum anti-
body reSponse in guinea pigs following intraperitoneal
inoculation. It was found that leptospiruria occurred in
all infected calves between P. I. days 13 and 20. The last

leptOSpiruria detected was on P. I. day 37.

Serum Antibodies

 

Specific serum antibodies against L. canicola were
first detected on day Six and reached a maximum between P.I.
days 17 and 21. The test was considered positive if ag-
glutination and/or lysis took place in the 1:100 serum

dilution, provided it increased on subsequent tests. In

26

this experiment positive agglutination-lysis was encountered
in dilutions of up to l:l,000,000. Results are summarized

in Table 9.

Post Mortem Findings

 

Carcass condition of four calves, 916, 920, 922, and
923, which were originally in excellent condition, were still
quite good, while the three others, 921, 924, and 925, were
in a rather poor condition and did not seem to have gained
weight after recovery.

Calf 922, which was killed on P. I. day Six Showed a
Slight congestion of the liver and kidney, and a slight in—
crease in the pericardial serous fluid. The suprascapular,
femoral, pOpliteal and mesenteric lymph nodes were slightly
enlarged and appeared edematous. One calf (921) had a
Slight endocarditis involving the mitral valve and calf 925
had some fibrinous pleural adhesions. Apart from these, no
gross lesions were evident except in the kidneys, which
showed greyish lesions varying from barely visible to about

two to three mm. in diameter.

Persistence of Leptospires in Tissues

Kidney tissues from five of seven infected calves were
shown by guinea pig inoculations to contain L, canicola. These
five calves were necrOpSied within 40 days following inocula—

tion; while guinea pigs inoculated from kidney tissues of

27

calves necropsied at 50 and 75 days and of control calves
were all negative.

Only one guinea pig inoculated with brain tissue
from calf 922, killed on P. I. day Six was positive in a
1:100 dilution of serum.

All guinea pigs inoculated with homogenized livers
and spleens from all calves were serologically negative,

Table 10.

Histopathologic Examination

 

The most extensive lesions were present in the kidneys
of calf 921 killed on P. I. day. 21. The renal lesions were
characteristically well demarcated and primarily in the
cortical region. The lesions were typified by intertubular
and periglomerular infiltration of predominantly lymphocytes.
In the affected areas the tubules were compressed and to a
degree replaced by the inflammatory cells. The glomeruli
were shrunken in some instances and Bowman's capsules were
thickened. Many of the tubules near the periphery of the
lesions were distended, Figures 10 and 11. All of the
remaining infected calves, including 922 killed on P. I. day
6, had minimal renal lesions of a type similar to but much
less extensive than calf 921, Figures 13 and 17. The control
calves had no renal lesions of consequence. There was one
small area of lymphocytes located perivascularly in the

cortical region of calf 918.

28

There were a few focal accumulations of lymphocytes and
neutrophils in the liver of 922 killed on P. I. day 6, Fig-
ures l4 and 15. In the regions of the portal triads of 920
and 922 there were greater numbers of lymphocytes than in the
control calves or in the remaining infected calves.

The medulla of one adrenal gland of 922, killed on
P. I. day 6, had an area of lymphocytic infiltration,

Figure 16.

There were intertubular accumulations of lymphocytes in
testicular sections of 921 killed on P. 1. day 21. These
lesions were not extensive or numerous, Figure 12.

No lesions were observed in the remaining tissues

examined in the infected or in the control animals.

DISCUSSION

A marked thermal response with temperatures ranging
between 104-107 F. and occurring during the acute phase of

the disease was reported in cattle with Leptospira pomona,

 

York, (1951), Gochenour (1953), Moore, eL_aL. (1956), and
Mitchell, eL_aL, (1960). Van der Hoeden (1955) had reported
high temperatures of up to 106 F. in one calf experimentally
infected with L. canicola. Similar reactions were seen in
sheep, Morse, _LaL. (1957), Lindqvist, __i_;___1_. (1958), and
in swine, Sleight, eL_aL, (1960), with L. pomona infections.
In this experiment, the temperature of the infected
calves started to rise as early as 14 hours after inoculation
and at the same time, leptospires were detected in the cir-
culating blood. This early leptOSpiremia with marked thermal
reSponse a few hours after inoculation is not common with
other leptOSpiral infections in cattle, York (1951) or with
.L. canicola infections in man and dogs, Bert6k, _L_al, (1961).
It seems reasonable to speculate that this outcome may be due
to one or more of the following factors. The comparatively
large inoculum of hamsters' blood taken at the height of their
infection may have had something to do with this. It will
be noticed that the experimental group that were infected

from the 16th and 28th hamster passage experienced the high

29

30

temperature peaks 24 hours before the first group, which was
infected with blood from the fourth passage. One may expect
enhancement of virulence to hamsters by repeated passage,
and hence more leptospires per unit volume of blood, Bertok,
333;. (1961).

On the other hand, this unusual reSponse might have
been induced because of incomplete adaptability of host and
parasite to one another. It was seen in this experiment that
L. canicola, killed hamsters in the first few passages
around the 4th day following their inoculation. This period
however, became gradually shorter and hamsters after the 10th
passage were killed in approximately 48 hours. It was also
noticed that calves inoculated with hamster's blood from the
later passages had more heightened response and an earlier
leptOSpiremia than the first group. These findings may
indicate an enhancement of virulence to cattle also, with
evident Shortening of the lag phase after inoculation. It
is postulated that this unusual reSponse may have been
reSponsible for the calves' ability to limit the progress of
the infection.

All other symptoms of anorexia, accelerated pulse,
lethargy and stiffness of legs were reported in the litera-
ture to occur during the acute phase of leptospirosis in

t.aL.

calves, York (1951), van der Hoeden (1955), and Moore,
(1956). In this experiment such symptoms could have been

caused by the fever alone.

31

The hemoglobinuria and jaundice that had been described
to occur in calves during the acute phase of natural infec—
tion with L. canicola, van der Hoeden (1955) and (1956) and
Turner, e§_aL, (1958), and also with_L. pomona, Reinhard
(1951), Gochenour (l953),and Mitchel,__§u_L. (1960), were
not encountered in any of the calves in this experiment.
Anemia, on the other hand, as a result of erythrocyte loss
as indicated by successive counts and reduction in hemo—
globin and PCV, apparently through hemolysis, was a common
finding in all infected calves, but the reductions were
not as great as those reported by Turner, _L'_L. (1958).

The fact that no isolations were made from liver and
spleen as early as P. 1. day six, indicated that leptospires

disappeared from these organs as early as they did from the

circulating blood. Leptospira canicola was shown to be pre—

 

sent in the brain on day six but no lesions either gross or
microscopic were seen in brain or meninges. It may, there—
fore be speculated that localization in the brain, which is
known to occur in humans, Dvoskin, eL_aL, (1956), does not
commonly occur in L, canicola infection in cattle.
Localization of L. canicola in the kidneys of cattle
and its subsequent shedding in the urine for sometime after
recovery, confirms van der Hoeden's findings (1955) and
(1956) and those of Turner, _L__L, (1958). The shortness of

the period of Shedding being up to P. I. day 37 as compared

to periods quoted for L, pomona, Morse, t l. (1957), was

32

evidently a result of the comparatively less extensive
localization in the kidney seen in this experiment. This
was evidenced by negative renal function tests and the
freedom of urine from Signs of nephritis, as can be seen
from Table 6 and Appendix 11. Apparently Serious damage to
the kidney tubules, that was reported by Low, _L_a}, (1956),
Gleiser (1956), and Arean (1962) with L. icterohaemorrhggiae,
did not occur in this experiment. This again could be attri-
buted to the exaggerated response of the host marked by a
high fever, early cellular response, early antibody produc-
tion, and probably a parallel tissue immunity that may have
limited the develOpment of lesions.

Results obtained from the liver function tests in the
seven infected and three control calves were not consistent
and were therefore difficult to interpret. All calves had
pre—inoculation clearance times comparable to the normals
described by Cornelius and Kaneko (1963). Apart from a
Slight delay seen between days three and six, most of the
calves had an increase in the 1/2 clearance time ranging
from .54 tollxx3 minutes and two of three control calves
showed a delay of 1.32 and 6.8.minutes respectively, Table 7
and Appendix 13. Except for this slight increase between
days three and six, most of the calves had a relatively
higher increase between P. I. days 13 and 20, and for the
controls on days 30 and 40 respectively.

It is surmized that the delay in clearance shown after

P. I. day 13 would not be accounted for by an inflammatory

33

hepatocellular injury that was described to occur in dogs

infected with L- icterohaemorrhagiae, Low, t l. (1956),

 

Gleiser, §L_al, (1957), or in guinea pigs, Arean (1962).
This is based on the minimal gross and microscopic hepatic
lesions, Figures 14 and 15, and on the finding that no
leptOSpireS could be demonstrated in the liver on P. I. day
Six, and were therefore assumed to have disappeared by the
end of leptOSpiremia, and the simultaneous appearance of
serum antibodies. Similar findings were reported with L,
pomona by Gochenour, _L__L, (1953), and Sleight, eL_al,
(1961).

It also appears that the relatively low hemolytic
qualities exhibited by L, canicola in this experiment, as
indicated by the comparatively less severe reductions in
blood values and the absence of hemoglobinuria and jaundice,
would not be expected to result in the severe hepatic de-
generation and centrilobular necrosis that were reported to
occur with L. pomona hemolysin in cattle and Sheep, Sleight,
£31. (1962).

Figures 14 and 15 are photomicrographs of sections
taken from the liver of calf 922 killed on P. 1. day six.
The only lesions seen consisted of focal accumulations of
neutrophils and lymphocytes. In the remaining experimental
calves these accumulations were either negligible or absent.

No signs of necrosis or disruption of hepatic cords were

seen in any of the calves.

34

Variable amounts of bromosulphalein (B.S.P.) were
recovered in the urine of all calves. Some workers have
Shown that the dye, following a single intravenous injection
is practically exclusively taken up by the liver and
excreted in the bile. Rosenthal and White (1925) found
that extirpation of the liver left the B.S.P. almost Lg
Lng_in the blood during the early period following injec—
tion. They also reported that if any, only traces are ex-
creted in the urine. Cantarow, _L._L. (1941), found that
practically 100% of the dye was removed from the blood by
the liver in the first 30 minutes. The same was reported

by Pratt, t 3;. (1952), and Cornelius and Wheat (1957),

who described extrahepatic excretion as insignificant.
Negligible amounts of B.S.P. in the urine of human

beings following Single intravenous injection was reported

t a}, (1960). Carbone, e£_§;, (1959), produced

by Wheeler,
experimental data Showing that small amounts of up to 2.7 i
1.1 mg/100 ml. are excreted in the urine of normal animals
while in others with hepatitis and obstructive conditions

of the liver, up to 26.2 :_5.1 mg/100 ml. were excreted in

the urine. Klein, §£_aL, (1933), on the other hand, claimed
that a portion of the dye is taken up by the reticuloendo—
thelial system, especially in the Spleen, a finding which was
also reported by Moses, eL_aL, (1948). Dogs with experimental
obstructive jaundice were Shown to excrete in the urine from
30 to 50% of the amounts of B.S.P. originally injected, Giges,

g: 3;. (1952).

35

Brauer, eL_31. (1955), found that substantial amounts
of B.S.P. were stored in various tissues following contin-
uous infusion, and that the kidney had 5.3% and the urine
from .2 to 1.7% of the amounts infused. He thought that
excretion in the urine was Sporadic and its occurrence
unpredictable.

In this experiment the B.S.P. recovered in one in-
stance was approximately 7.6 mg. in a 62 ml. urine sample.
This might not have been the total amount in the bladder at
the time of collection. There was no evidence of renal in-
sufficiency or of serious hepatic dysfunction at the time
the samples were taken as indicated by the various tests.
In view of this, it is Speculated that cattle may normally
excrete in the urine, amounts of B.S.P. that may affect the
sensitivity of the test in this Species. More controlled
experimental work is definitely needed to determine the role
of the kidney in the excretion of B.S.P. in cattle.

Slight initial leukocytosis, which was in some in—
stances followed by a Slight leukopenia, had been reported
in various animal hosts and man, York, t al. (1951);

Roberts, 3331. (1952); Lindqvist, e 1. (1958); Sleight,

3131. (1960); and Berto’k, §£__1. (1961).

In this experiment, there was a noticeable leukocyto-
sis amounting to about 160% of the pre-inoculation values in
some calves, followed by a total leukopenia with up to 55%

of the leukocytes disappearing, Figure 5. Of interest,

36

however, was the marked neutrophilia which reached about
400% in one case and averaged 230% of the pre-inoculation
values for the seven infected calves during the first two
to three days, Figure 6, Appendix 6. Once more the neutro—
penia was quite marked after postinoculation day five. It
will also be noticed that the extremes of neutrOphilia and
neutropenia were encountered in the calves receiving the
higher passage inoculum. This might be interpreted as
either a true defense response against the increasing number
of leptOSpires, or as a result of the febrile condition in
calves. I

Another interesting feature of the animal reSponse
was the monocytosis that was seen in all the infected
calves and, to a certain extent, in the control calves. In
some infected calves the monocytes reached over 20% on cer-
tain days during the leukOpenia; but Since a Slight mono—
cytosis was evidenced in control calves, it might be sug—
gestive that at least in part, this could be a reSponse to
some factor in the hamsters' blood, Figure 8, Appendix 9.

Absolute lymphOpenia was seen both during the initial
leukocytosis and the subsequent leukOpenia. Lymphocytes
have been reported as part of the reaction in various tissues,
1. e., kidney, in leptospiral infection. The only correla-
tion between a similar lymphocytic infiltration of some
tissues in this experiment Figures 10 through 17, and the
number of circulating lymphocytes was a relative lymphocyto-

sis, Appendix 8.

37

One calf, 921, killed on P. I. day 21 had the most
extensive kidney lesions. These consisted of intertubular
and periglomerular infiltrations, predominantly by lympho-
cytes. Tubular and, to a lesser extent, glomerular degener-
ative changes were seen in the affected areas primarily in
the cortex, Figures 10 and 11. Renal lesions of the remain-
ing calves were similarly typified but much less extensive,
Figures 13 and 17. These findings Seem to be in conformity
with the Short period of localization of L, canicola in the
kidney and the short period of leptoSpiruria, seen in this
experiment.

Intertubular accumulations of lymphocytes were seen
in testicular sections of calf 921, killed on P. I. day 21,
Figure 12. These were not extensive but they may, however,
suggest a tendency of L, canicola to localize in testicular
tissue. The.extent and significance of this in connection
with transmission by coitus may only be verified by further
experimental work with adult bulls. Similar findings were
reported to occur in male cattle experimentally infected
with L, pomona, Atallah (1963).

A11 calves had a marked antibody response against L.
canicola and their sera were positive in dilutions ranging
from 101 to 106. Weak positives in dilutions of 1:10 were
first noticed on P. 1. day five. These were not considered
significant until day Six when positive agglutination—lysis

reactions were obtained in serum dilutions of 1:100 and more,

38

reaching a maximum around the second to third week after
infection. Similar findings were reported before with L.
pomona infections, Moore, §L_§L. (1956); Boulanger(1958);
and Steele (1958).

The immune response in all infected calves is also
comparable with the early thermal and cellular responses;
antibodies having been detected as early as P. I. day five
and reaching a maximum also quite early. This may further
explain the termination of leptOSpiremia by P. I. day five

and the short duration of renal localization.

SUMMARY AND CONCLUSIONS

An experiment was conducted on ten calves, seven of

which were infected with L, canicola, to obtain valid ex—

perimental data on infection in cattle caused by this

serotype.

The following were considered to be the most

pertinent aspects of the experimentally produced disease.

1.

An initial acute febrile stage of Short duration,
characterized by a high temperature and other
accompanying symptoms.

Anemia with moderate reduction in blood values
occurred, but there was neither hemoglobinuria
nor jaundice.

LeptOSpiremia was evidenced between P. 1. days
one to five followed by clinical recovery.
Cellular reSponse was evidenced by initial
leukocytosis and neutr0philia followed by a
total leukopenia with relative lymphocytosis.
Humoral antibody reSponse starting day six and
reaching a maximum between days 12 to 21 with
titers up to 106.

Evidence of localization in the kidney with
subsequent Shedding in the urine for 37 days as

determined by guinea pig inoculations.

39

40

7. There was no impairment of renal function and

only a slight impairment to liver function.

8. Gross and microscopic lesions were practically

confined to the kidney.

The experiment confirms earlier findings by van der
Hoeden, Turner, _L_aL, and others, that L. canicola can
establish itself in cattle; and, although clinical recovery
took place promptly in all infected calves in this experi-
ment, yet the apparent retardation to growth was quite sub—
stantial. Valuable experimental data on the course of
infection, fate of L, canicola in cattle and the behavior of
the latter as host Species, were gained. This was definitely
needed to remove, in part, the deficiency in the literature
concerning this serotype in cattle.

Moreover, localization in the kidneys with subsequent
shedding in the urine, increases the hazard to man, cattle,

and other animal species, if proper precautions are not

taken.

41

TABLE l.—-Summary of Leptospiremia,Leptospiruria, and
Serum Antibody in Infected Calves

 

 

Postinoculation Days

 

First End First End First Maximum
Calf Lepto— Lepto— Lepto- Lepto— Serum Serum
No. Spiremia Spiremia Spiruria Spiruria Antibody Antibody

 

924 2 4 17 37 6 20
925 2 4 20 28 5 20
923 2 1+ 20 32 6 17
916 2 4 14 up to 30 6 21
921 1 11 14 up to 21 6 21
920 l 4 13 up to 14 6 11
922 2 5

 

42

TABLE 2.--Summary of Reductions in Hematologic
Experimental Calves

Values of

 

 

 

 

Average Lowest
Calf Values Values Values Amount Percentage
No. Tested A.I.** P.I.*** Reduction Reduction
915* Hb.° 12.00 11.60 .40 3.3
P.C.V. 32.80_ 32.00 .80 2.4
R.B.c.x103 10056 9340 716 7.1
924 Hb. 11.80 8.40 3.40 28.8
P.C.V. 33.60 22.00 11.60 34.5
R.B.C.x103 9685 6940 2745 28.2
925 .Hb. 12.20 9.20 3.00 24.6
P.C.V. 32.50 25.00 7.50 23.0
R.B.c.x103 8860 6815 2045 23.0
918* lib. 10.40 9.60 .80 7.7
P.C.V. 30.50 28.50 2.00 6.5
R.B.c.x103 7815 7220 595 7.6
923 Hb. 11 60 10.10 1.50 13.0
P.C.V. 3 33.00 27.00 6.00 18.1
R.B.C.x10 8745 7000 1745 20
916 Hb. 12.5 9.00 3.50 28.0
P.C.V. 3 35.0 25.00 10.00 28.5
R.B.Cx10 8510 6100 2410 28.3
919* lfla. 10.30 10.20 .10 1.0
P.C.V. 30.00 28.50 1.50 5.0
R.B.C.xlO3 8180 7640 540 6.6
921 Hb. 10.30 8.10 2.20 21.3
P.C.V. 30.00 24.50 5.50 18.3
R.B.C.x103 8570 6 80 2490 29,0
920 Hb. 13.50 9.70 2.80 20.7
P.C.V. 36.50 27.00 9.50 26.0
R.B.C.xlO3 8720 6250 2470 28,0
922 Hb. 11.30 10.00 1.30 11.5
P-C-V. 3 33.00 26.00 7.00 21.2
R.B.C.x10 7840 6870 970 12.3
* Control calves oHb. gm. hemoglobin/100 ml.

Ante-inoculation blood

Post-inoculation

** A.I. =

*** P.I.

43

mm>Hmo Hoppcoo*

 

O O O O O O O O O O O O O O I O O O O O O O O I O O +
O O O O O O O O O I O O O O O O O O O O + O O O +
O O O O O O O I O O O O O O I O O O I O O O O + +

... ... ... ... ... imam
... ... ... ... ... leaa
... ... ... ... ... imam
+ ... ... ... ... mam
+ + + ... ... 333
+ + ... ... ... cam
... ... ... ... ... cum
... ... ... ... ... mam
... ... ... ... ... mam

co. .0. on. co. .00 :Nm

 

ON

SH ea me me we ow m m s 6

Ln
:7-

m
(\J

H .02
mamo

 

mama coHpmHSoochmom

 

mo>amo eopoomcH mo oCHLD
CH popmppmcoEom mm: pooam pHSooo Loagz co mama

COHpmHSOOQHLmomII.m mqmde

44

 

 

 

 

 

 

 

Hm.afl me.afl Hm.aH em.afl mo.afl HH.AH we.afl om.&H me.HHU.mexncoHH
-Hwa nHHoo

moe.w mee.w mem.m Hmm.m mam.w magma wmm.m wem.ml. Hmw.m eooam pom

om.MH om.mH o:.aH oo.mH oe.mfl om.afl oo.mH mm.aH om.afl casHo>
Hebe

oo.mm om.Hm mm.em mo.om mm.em mm.am mm.mm mm.em oH.Hm eoxoom

omafl oo.dH msafl sax“ swam mmafl ms H. mm.H mmaH.HsooHn.sm
owe

os.oH om.oH mo.HH ms.ofi wo.HH mm.oH om.HH mm.oH om.oH -oamoaom

msafl mew memfl ooafl mmnfl mmsfl ewafl Hmafl defl .me

\newso

mam mme oom one mm: mom omm mom eom -ocwnom

mmfl manfl mmmfl HHmH mwafl mmMH Hmmfl msdfl modfl .ee

_ \mmpho

mos emoH mmm mew mom new mmw mos How -ocoz
momdfl modem comm mam“ mmeafl 26mm emsfl mash mam“ .me»
\mop o

mmos omms mmmm mmos mews oamm some some Heme -osoesq
mamafl :wmaw 6mm“ samfl mmmAH oomafl wmmfl HsmH mmmfl \.me x
n we

mwmm omom moom essm mam: ems: osmm mwmm mmoe -osesoz

 

 

oomfl meow page“ mmeafl mmwfl momfl mam“ meow mmmfl max nooso

logsmq

Homes mmeflfl omefie emcee mwmme memmfl wmmee mwmee mmmfifi Hmooe

mH HH 0 S m m m H .HSOoCH mQOuomm
10am

 

coapmazoocflpmom mama

 

mo>amo Hoppcoo mo mmzam> oawoaoumEmm

ecm mussoo opzooxsmq medaomn< one go Amv coauma>mm egmecmpm new hwy smwznn.: mqm<e

45

 

 

 

 

 

 

 

 

 

mm.H. oe.H. meafl emafl omafl mSaH oo.aH somafl mmmfl .ma\wsowe
IHHE maaoo

meo.s mSH.s Seo.s mom.m wam.m mmm.s Ham.» mam.w eos.m pooem pom

om.mfl oo.mH om.MH om.mH ss.aH oo.mfl oo.aH mm.mfl we.mfl oaaeo>
Heme

om.wm mm.sm oo.mm om.mm ms.sm om.mm om.mm m:.mm sm.mm bosons
mmafl osaH oo.aH mmafl omafl mmafl owaH owafl oo.aH.HeooH\.em
Canoaw

ms.m oo.oH ow.m sm.m ow.m me.oH om.oH mm.HH ww.HH -oEom
ommfl Roam mow mm“ meMH oaafl mmmfl swam scam me o
new:

was She one ewe wee emm mmm rem msm -ocwnom
wmafl emfl osafl mHMH mmofl mmnfl Hemfl mmmfl memfl .mss
new 0

mam see was mos some mmm msm mam com -osoz
mamafl eflmfl mmmfl Hose“ omeafl oemaw smomfl mamaw mmmfl .meS
, \mwp o

smom Hmem swom Hmem mmw: mmee new: ream wwmw -osoesu
Raw was“ man Sow coin mean swans SSH Emu Mime n
new:

mmmm mmmfi come mama mmem Hmwm mmmm meow Hmmm -owwsoz
oHeHH. madam mHmAH osmmfl mmmmfl wmmmfl smmmfl mmmmfl osmAH .me»
\mop o

mmmm oHom sows Hoes smsw mmeea mmmee omeoH mmHoH -oxsoq
me He 0 s m m m H .HsoocH nwoeobm

mam

 

COHpmasoochmom mama

 

 

 

mm>Hmo couoom2H wo modam>

oesoossoq wawcowoeewm oeseonea one so A

O
.(

oawOHOmemm cam mpssoo
v cowonw>om ewwesowm one hmv sews--.m mamas

46

TABLE 6.--Summary of Renal Function TestvMean Values of B.
U. N?*Before and After Infection, in mg/100 ml. of Blood

 

 

 

Maximum Minimum Day of

Mean for Mean for Level Post- Maximum
Number Pre—Inoc. Postinoc. Postinoc. inoc. Level
924 20.50 17.00 22.50 10.00 2
925 17.00 14.10 18.00 8.00 37
923 18.75 15.30 17.50 11.00 1,4,5,20
916 14.00 14.60 16.50 11.50 20
920 12.50 14.60 17.50 12.50 :3
921 12.50 17.30 21.00 12.50 3
922 13.20 16.00 20.00 13.00 4
915* 11.75 13.30 16.50 8.00 9
918* 16.50 17.70 23.00 10.00 8,9
919* 14.00 12.80 17 00 9.00 1

 

*Control calves
**B.U.N. = Blood Urea Nitrogen

47

TABLE 7.-—Summary of Liver Function Test;Mean, Pre— and Post—
inoculation Half Times for Clearance of Bromsulphalein in

 

 

Minutes
Mean Mean Maximum P.I. Day
132? SEEMS SEEMS; liniiiiiis 01.24.3222?
(m) (m)
924 3.68 4.05 2.64 20
925 4.32 4.83 2.00 20
923 2.13 3.01 1.63 17
916 3.99 4.38 2.71 14
921 4.58 4.21 .63 3
920 3.45 4.73 4.07 14
922 3.75 4.36 .54 5
915* 3.94 4.94 1.32 13
918* 5.18 5.68 6.80 40
919* 4.08 3.40 .27 3

 

*Control calves
m = minutes

48

TABLE 8.--Duration of LeptOSpiruria in Experimental Calves
Confirmed by Guinea Pig Inoculations

 

 

Post-

inoc. Days Postinoculation

 

Days 915* 924 925 918* 923 916 919* 921 920 922

 

1-12

13 ... ... ... ... ... ... +
14 ... ... + ... + +
17 ... + ... ... ... + ... +

20 ... + + ... +

21 ... +

24 ... + + ... + +

27 ... ... +

28 ... ... + ... +

30 ... + ... ... ... +

32 ... ... ... +

37 ... +

40

43

SO

63 ...

75 ...

 

+ = Positive agglutination-lysis in serum dilutions of
1:100 or more.
* Control animals

TABLE 9.——Antibody Titers** for L.

49

Infected CaIves

Canicola in Sera of

 

 

 

 

Post- Calves
inoc.
Day 915* 924 925 918* 923 916 921 920 922
1-5
6 +2 +2 +2 +2 +2 +
7 +2 +2 +2 +1 +3 +3
8 +2 +3 +4 +3 +4 +5
9 +2 +3 +4 +3 +4 +4
10 +2 +3 +5 +3 +4 +5
11 +3 +3 +5 +4 +4 +4
12 +3 +3 +5 +4 +4 +4
13 +3 +3 +5 +4 +4 +4
14 +3 +4 +5 +4 +4 +4
17 +3 +4 +6 +4 +4
20 +4 +5 +5 +4
21 +6 +4
24 +4 +5
27 +4 +5 +4 +4
28 +4
30 +4 +4 +4
32 +4 +4
37 +4 +4
40 +4 +3

50

TABLE 9.-—Continued

 

 

 

 

Post- Calves

inoc.
Day 915* 924 925 918* 923 916 919* 921 920 922

 

 

43 ... +3 +4
50 ... ... +3
63 ... +3
75 ... +3

Killed 75 75 50 4O 4O 30 21 21 14 6

 

*Control calves
**The titers are expressed as the exponents of the highest ten-

fold serial serum dilution where agglutination—lysis
occurred.

TABLE lO.--Summary of Persistence of L. Canicola in Tissues

of Infected Calves

 

 

 

Case Days Post-
No. Kidney Liver Spleen Brain inoculation
915* 75
924 75
925 50
918* 40
923 + 40
916 + 30
919* . . 21
921 + 21
920 + 14
922 + + 6

 

*Control calves

+Positive by guinea pig inoculations. Agglutination and/or
lysis of L.
dilutions of 1:100 or more was considered positive.

canicola antigen by the guinea pigs sera in

Body Temperature F

107~~

106+

I

105‘

104--

103‘t

T

1011

 

102--
1A\\ /.\\ ) \ /

52

 

Infected

 

---- Controls

 

 

4 L 1 1 1 1 1 1 l 1 1
I I 1

1234567 891011121314
Postinoculation Days

Figure l.—-Mean Values of Daily Temperatures

of Experimental Calves

Body Temperature F

 

 

 

 

 

 

lO7-P Infected
---- Controls
106-—
105*“
104+
103“
1021-
(A\ /.\ ’ \ /
\ / x ./
V V —-'-—0
101--
1 i t : 11 i : : + L +4 5 + 11
123456789101112134

Postinoculation Days

Figure l.—-Mean Values of Daily Temperatures

of Experimental Calves

Hemoglobin—-Percent of Pre—inoculation Levels

130

120

110

100

90

80

70

6O

53

 

 

Infected
-—-—- Controls
.A\1 .;~13 A Pre-inoc.
r f I \
V ‘r’AV ‘7‘fi-V’k“

 

 

l

131117171711'fi‘1
1234567891011121314
Postinoculation Days

Figure 2.--Mean Hemoglobin Levels of Experi—

mental Calves, Expressed as Percent of
Pre-inoculation Values

Percent of Pre—inoculation Values

110

100

90

8O

7O

60

54

 

 

 

 

 

Infected
—--— Controls
as
L’A\v"\ {A A Pre—inoc.
—. ‘e\ If \w-At‘t‘ 1‘.
\r
a.
“'(b
12 3 4 567 891011121314
Postinoculation Days

Figure 3.--Packed Cell Volume; Daily Mean

Values, Expressed as Percent of Pre-
inoculation Levels

Erythrocytes as Percent of Pre—inoculation Counts

120

110

100

90

8O

7O

60

55

 

Infected

---"—Controls

Pre-inoc.

 

 

 

 

1
::+::++S:;++#:
1234567891011121314
t

Pos inoculation Days

Figure 4.--Mean Values of Daily Erycthrocyte

Counts Expressed as Percent of Pre-inocu-
lation Levels

Leukocytes as Percent of Pre—inoculation Counts

56

 

 

 

-————— Infected
—--_- Controls
120*-
A
/\
110+ // \
/ \/ '\
-( I \ x
l/ ‘ *V/ \ Pre—inoc
100 \ 1’ L '
\-—.—-’ \\
904+
80+
70“.
t f 4+ i : t* i i i +5 i i i :-i
12 3 4 5 6 7 8 9101112131415
Postinoculation Days

Figure 5.—-Mean Values of Daily Leukocyte Counts
of Experimental Calves, Expressed as Percent-
age of Pre—inoculation Levels

Total Neutrophils—-Percent of Pre—inoculation Values

240
230
220

210
200

190
180
170
160
150
140
130
120 -
110 -
100
90*
80+
70;
60+
50*
40 «
30 -
20 -

10 -r-

   
   
  

I

F

El"

 

57

Controls
Infected

 

Pre-inoc.

4L 1 1 1

l l I L

 

l 234567891011121314

Postinoculation Days

Figure 6.--Daily Mean Values for Total NeutrOphilS
Expressed as Percentages of Pre—inoculation

Values

Lymphocytes as Percent of Pre-inoculation Values

58

 

 

 

Infected
___._ Controls
120‘7
rA\
/ \
1 / \
llO‘L I\ X ’ \\
’1 1‘ ,\ I '0
f‘ ’\ /\ /
l \ [\I \I
100 1 l *1 Pre-inoc.
\ l
\l
/
\/
904- V
80-~
70.4;
60‘)
i 5 i i i : + i i 1 t % *tet ~
1234567 891011121314
Postinoculation Days
Figure 7.-—Daily Mean Values of Lymphocyte

 

Counts Expressed as Percentages of Pre—
inoculation Values

MonocyteS-—Percent Increase and Decrease

230 ..
220
210
200
190
180
170
160
150
140
130
120
110
100
90
80 ‘r
70 4'
60 --
50 4.
4o --
30 *-
20 «L
10

-—————-Infected

-u——-Controls

 

 

L l 4 1 I

1 A l
f T 1 ‘I

7 8 9 10 111213 '14

J 1
V l

5 6
Postinoculation Days

Figure 8.--Showing Daily Mean Values of Monocytes
Expressed as Percent of Pre-inoculation Counts

l—‘w-

234

Pre-inoc.

EosinophilS-—Percent Increase and Decrease

220+
210+
200"
190»
180--

170+
160-
150.
140-
130+
120+
110+

T I

I

60

Infected

 

x _____ Controls

 

100
90+
80+
'70--
604-
50+

40--

20-
10‘

U

 

Pre—inoc.

 

I l l
I

 

4567§39mflfiuwfl
Postinoculation Days

2' 3

Figure 9.-—Eosin0phils Mean Values of Daily

Counts, Expressed as Percentages of Pre-

inoculation Levels

 

Figure lO.--Kidney Section, Calf 921.

   

\ ‘\ ‘ § '
u D” " '
,5 '0 ~
‘0‘ ’
i! l
. i d ,

Killed on P.I. day

21 to Show (A) intertubular infiltration, (B) periglom-

erular

infiltration predominantly by lymphocytes, (C)

shrunken glomerular tuft, (D) dilated tubule. x 187.

{
l
l
l
i
I

 

 

 

Figure ll.--Kidney Section, Calf 921.

Note (A) thickened

Bowmans capsule, (B) predominance of lymphocytes in

periglomerular region. x 750.

 

 

 

Figure l2.--Testicle, Calf 921. Note interstitial infil-
tration predominantly by lymphocytes. x 187.

 

 

 

 

Figure l3.--Kidney Section, Calf 922. Killed on P.I. day
6 with intertubular infiltration by lymphocytes. x 187.

 

 

 

Figure l4.--Liver Section, Calf 922. To Show focal areas
of lymphocytic infiltration. x 187.

 

 

 

 

 

 

 

Figure l5.--Liver Section, Calf 922. Note lymphocytic
infiltration in the region of portal triads. x 187.

 

 

 

Figure l6.--Medulla of Adrenal Gland, Calf 922. With focal
infiltration predominantly by lymphocytes. x 187.

 

 

 

 

Figure 17.--Kidney Section, Calf 923. Killed on P.I. day
40. Typical intertubular lymphocytic infiltration. x 187.

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APPENDICES

74

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00.0 00.0 00.0 0H0 H00 00.0 00 00.0 00.0 00.2 0.0
000 000 H00 0H0 0H0 000 0H0 000 000 0H0 0000
HthCOO HthCOO HthCOO

 

.WE\©oH x mpcsoo memoOanhem 0:0 mngEzz mm>Hmo

 

 

mm>Hmo HmpCmEHemme 0o mpczoo mpzoohcphemnu.0 mezmmm<

79

 

 

 

 

0000 0000H 0
0000H 0000 0HO0H 00
0000H H000 0000H 00
0H00 000HH 00H0H 00H0 00
0000 0000 0000H 00
HHO0H 0000H 0H00H 0000 0000 00
0000H 0000 0000 0000H 00
0000H 0000H 00H0H 000HH 00H0 0000 000HH H0 w
0000 0000 0000 0000H 00H0 H000 0HOHH 0H 3
H000 0H00 0H00H 0000H 000H 000HH 000HH 0000H 0000H 0H 1
H000 0HH0 0000H 0000 000 0000H 0000 0000 000HH 0H w
H000 0H00 0000H 0000 0000 0000H 00H0 0H00 0000H 0H m
0000 0000 0000H H000 0000 0000H 0000 0000 000HH HH 1
0000 0000 000HH 0000 00H0 0000H 0H00 0000 000HH 0H m
0000 0000 0000H 0000 0000 0000 0000 00H0 H00HH 0 W
0000 0000 0000 0000 0H00 0000H 0000 0000 0000H 0 u
00H0 0000 0000H 0000 0000 00HHH H000 0000 000HH 0
0000 H000 00H0 0000H 0000 0000 0HOHH 0H00 0000 0000H 0
0000 0000 H000 00H0H 0000H H000H 000HH 0000 0000 0000H 0
0000 000HH 0000 0000H H000H 0000H 000HH 0000 0000 000HH 0
000HH 0000H 00HHH 0000H 0000H 0000H 000HH 0000 0000 000HH 0
00HHH 0000H 000HH 0000H 00HOH 00H0H 000HH 0000 0000 0HO0H 0
0000H 00HHH 0000 H000H 0000H 0000H 000HH 0000 0000 00H0H H
T..d
000HH 0H00H 00H0 000HH 000HH 0000 0000H 0H00H 0000 00HHH wnw
U _
000 000 H00 0H0 0H0 000 0H0 000 000 0H0 0000
HOLQCOO HOQQACOO HthCOU

 

.WE\0pczoo wpmooxsmq H0008 0:0 0009552 00>H0o

 

 

00>H00 H0000eH000x0 0o 0000o0 000oo0000 H00o0 0HH00--.0 0H02000<

80

 

 

 

 

0000 0000 00
0000 000H 0000 00
0H00 0000 0000 00
00HH 0000 0000 000H 00
0000 0000 0000 00
0000 00H0 H00H 0000 000H 00
0000 0H0H 0000 00
00H0 0H00 0H00 000H 00H0 0000 000H 0000 H0 .0
0000 0000 H000 0000 000H 0000 0H m
000H 00HH 00H0 0000 0000 0HOH 0000 000H 0000 0H NH
000H 0000 0H00 00H0 0000 000H 00H0 H00 0000 0H u
000H 000H 0HH0 0000 0H0H 000H 0000 000 H000 0H m
000 000H 0000 000H 0000 000H H0H0 00HH 0000 HH m
0H0H 00HH 0000 000H HHOH 000H 0000 000H H000 0H 9
000H 0H0H 00H0 000H 0000 000H H00H 00H0 0HH0 0 “H
00HH 000 H000 000H 000H 0000 0H00 000H 0000 0 m
000H 00 0000 000H 0000 H000 00H0 0H0 0000 0
0000 0000 000 0000 000H 000H 00H0 000 HH0 H000 0
0000 0000 000 0000 0000 0000 0000 000H 0H00 0000 0
0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0
0000 0000 0000 00H0 0H00 0000 0000 0000 HO0H 0000 0
0H00 0000 00H0 0000 0000 0000 H0H0 00H0 000H 0000 0
0000 0000 000H 0000 0000 00H0 0000 0000 00HH 0000 H
0.0.
0000 0H00 HO0H 0000 H000 0H00 0000 0000 000H H000 w.w
000 000 H00 0H0 0H0 000 0H0 000 000 0H0 0000
Hogpcoo 0000000 Hoppcoo

 

.WE\000000 2000000002 000:0Emmm 000 0000852 00>H0o

 

 

00>H0o H0020EHgmmxm 00 000000

HH00o0000z 000005000 0HH00--.0 0H02000<

81

 

 

 

 

on. co. co. om
... 00 00H 00
... 00H ... ... 00
co. co. Hom \Lm
owl—.1. MUCH .0. 0.. 0w ... om
... ... 00 000 00
... H0H ... ... ... ... 00 ... H0
... ... mm ... mOH ... WWW wHH NH M
00 ... 0H0 000 ... ... ... ... ... 0H 0.
... ... NOW :0 ... ... mm MN ... MH I
... 00H 00H 0 00 ... ... 00H 000 0H w
... 00H 00H 000 ... ... ... 00 0HH HH m
... ow QQH Hm ... ma mm ... OH I
... 00 H00 0H0 000 00H ... 00 000 0 w
... ... ... 3w 3mm ... wmm ... ... w m
... Hm ... ... mmfl ... ... ... ... mHH N U
QQ qu mm ... MQH ow ... ... ... WNW m
... ... ... NMH MJMH mom @HH ... mwfi ... m
00 000 00H ... 000 00H HHH ... ... ... 0
000 000 ... 000 000 000 000 000 ... 0HH 0
000 HHH 0H0 ... HO0H HOH ... 00H 00 00H 0
00 0HH 00 00H ... ... 000 00 ... ... H
0:0
.0. wu—HN HNL mp: 000 000 0.0 $3“ ow mmm wnc
000 000 H00 0H0 0H0 000 0H0 000 000 0H0 0000
0000000 0000000 0000000

 

.mE\000000 0000000302 000000000I002 000 0000052 00>000

 

 

 

000000 H000000302 000000000-cOz--.0 00020000

82

 

 

 

 

mqmm mozw m»
moww maom mwmm om
ommm me: :map m:
mom: pomp mwwm Hwom o:
wqwm mom: Hmfim Fm
wmzp mono Hflpm muam wmmm om
mmm: wa: moon um
swam :mmm memo maze mmaw comm mwmm omwm Hm
mfimm :mmm Name mmwm wmmm ammo NH M
mam: mmqm :mmm mzam mzwm mmmm bmmw mmpw mmam 3H m.
Hmmm ozqm swam omfim momm mama mmmm mqmm :mom ma I.
ommm mmmm ammo momm momm mmqm mmwm mmmm mmfim NH w
mmfim mzwm mqmm mam mwmm Hmmm mmom owmm mmmm HH m
:Hmm moo: mmwm mom wmmm omooa mmam mflmm pmmm OH I
omom bzpm ammo Hmmm mam: mmom mmflm mqmm mom» m m.
momm :mmm wfimm ammo mamm mama mmwm mmpm wmfiw m m
mowm :mmm mmmo mmwm pmmo ommw mflmm mmwm mHH» w u
mmmm wpam :mwfi mmwm ammo mqmm Noam ommm :mo» mzow w
mmm: mwaq Hmmfi mmmm mwmm mmwm mflmp mmmm mwmm “mmm m
mmmm mom: mmmfl mmmm mom» mwom mqmm mpm: wawm mwmm :
mmwm mmmm mow: mwmm mmmo mmpm Hwom wmaq mwmm mwmm m
mama wmm» mmmm Hawm mom: flame mmqw azam swam mwmm m
mmmm @535 awe: omom mwm~ ommw mmmm ammo mzmm mmqw H
T.. .d
$8 3% E: 8: mm? is 8mm. mmow 3% 3% m m
mmm 0mm Hmm mam mam mmm mam mmm 3mm mfim mxmm
HOFHPQOO HOchCOO HOQHVCOO

 

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83

 

 

 

 

:mm mum mm
mHz mHo mpw om
:om :Hm on m:
Ham mmm mom mwm 0:
3mm MH: mmmH mm
mHmH mmm mmp mzw mmm om
mmm mam mmm pm
mom mom NomH mNHH mmHH mwm Nmm mm: Hm a
Ham Hm: wnm mm: mmm m»: NH m
HHN mm: mqm moo mmm Ham mom pHm mom :H m
mom mwm wa mmm mmm Haw mam mm: mm: mH u
mwm pm: woo woo mwm :mHH om: mmm mow mH m
mow Hmm omoH 0mm mom mmmH mmo mom mam HH m
mmN wmm :ww mam mp0 mmw map mow mom OH 2
mm ppm NmmH nmoH oHp mmN mop mm» co: m n”
wqm mam :on omm Nam mqm mmm Hmo :mm m m
mm» mp5 omoH wHoH mmm mow mum :mm om» w
omoH mmoH Hzm com www mmw mNm mqu mww Hm» m
mwwH mmmH mom mmm mmmm mmmH 3H» Nam mHHH mmoH m
mmm mpm Nam mmm mmmH mmHm mom mum @mHH HzmH :
mm :3@ mm» mwm mwm 5mm omHH me won Ham m
mp mmm mmm mon mom mom Hm: Nmm mmm :mm m
mwm mm: Hm: 35w 0mm mmm mmm mm: :mm mm@ H
mm
0mm :mw Fm: ow: :mm mmm mmm mHmH Hm: mom w.w
mmm 0mm Hmm mHm @Hm mmm mHm mmm :mm mHm mHmm
HOchCOO HOLUHQOO HOLHCOU

 

.me\mpcsoo mphoocoz UQm mpwpesz mm>Hmo

 

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mm: 33 E
mHm ms: Nam om
zoo com com m:
momH mp: mom mwm o:
mmm mmm ... pm
wmm mm» mmm me HmoH om
wmm mzm mpm mm
mmH mom mam me 2mm mpm cop mam Hm a
mmo mwm Hm: mm: mm» mHH NH 0
mow ... on mom How mam ms: pmm HwH :H m.
mH: mm HHH Hmm mqm omH wwm «Hm :HH mH W
Hzm mmH mmm mm mom mwm @wm :om me NH 0
mmH ... moH mwm Hmm sz mm mmm omm HH m
mp omH mmm Hm Hm Hmm ... mom ... 0H m
mmm mpH mm: mm 05 Nam NNH me ... m 1
moH om Ham ... ... mHm mm owH ... w m
mmH Hm oqm mm Hm Hm» mm mmm ... N u
mow m3: mm :Hm zaH om mmm 5» 5mm ... o
How mm no How :mH ... pom ... mum mmH m
0mm ... Na mmm mmH ... mmm ... mm mmm :
mHHH HmH HHH mom ... qu Hon ... mm: mHH m
owm mam ... Hon :m: HmH mam omH mm: moH m
mon wqu Hm: mpm NH: Hmm mom mm on ::m H
T. .d
0mm mmm 3mm HHH mmm mmm omm mmH Ho: mHH w a
U _
mmm 0mm Hmm mHm mHm mmm mHm mmm 3mm mHm mmmm
HOcHanOO HthCOU HOchQOO

 

.@E\mpcsoo HHSroHmom Cam mprESZ mm>Hmo

 

 

mw>Hmo HmemEprme mo macsoo HHQroHmom hfiawaun.oa XHQmem<

APPENDIX ll.——Resultscfi?lhfijmeExamination Showing pH and Specific Gravities

 

 

Calves Numbers With pH* and S.G.**

 

Control

Control

Control

915 924 925 918 923 916 919 921 920 922

Days

 

 

 

\Q m CU :I‘ Lfl (I) Ln
m m m m m m m
0c) Odo :ro CDO 0J0 CDO CDO
GDri GDH QDH GDH QDH GDH QDH
O N m O 0 L0 CO LO (\1
m m m :r m m (\J m m
OCD r*O "TO JWO 0J0 0J0 r40 OJO NDO
GDri GDH QDH QDH QDH QDH QDH d)H bqa
LG (I) KO (1) :1“ :1” O H CD
m m m (\J m m m m m
r40 (“CD C“C> :TC) :ro 0J0 U\O :‘O ”10
Glri GDH QDH QDH GDH GDH QDH QDH NMH
U\ N— K\ O -: CD Ln (fl L0
00 m m m m LI\C\I m m (‘0
CO NO NO NO NO HO :I'O NO HO
GDri QDH GDH QDH KDH GDH GDH QDH me
K) F— H W\ KO \0 b- O O
#4 N 3‘ W3 W3 N N N3 N1
rHC) K30 :r0 030 b—O KDO CDO W30 O\O
dDri GDH QDH GDH QDH b—H d) GDH b—H
K) -d' O F— K\ H m\ N O
N m :1” m m m m m on
(DC) CDC b~© d)O CNO r40 GDO K\O :‘O
(IDH (DH [\H b—H [\H QDH [\H [\H [\H
C) K\ O N N H O E\ (D
Lf\H m m m m m m m (\l
EHC) UNO U\O b—O b—O b—O KDO GDO O\O
GDP! GDH GDH QDH GDH GDH dDH QDH b—H
o N O GO 00 N O O CO
W1 “3 “l N N N “3 N H
QC) b—O 010 W30 r40 C30 C30 (DO C30
GDri GDH GDH GDH GDH GDH QDH b—H QDH
C) O N H O F— H O F-
m m m m m m m m 01
4'0 KOCD -#CD (“CD (DC) P~O 0J0 #40 3‘0
QDri GDH QDH GDH QDH GDH GDH QDH QDH
O C) U\ a) d) O O H O
(\J (\J H H H (\J (\J H H
r4C> WWO :r0 ()0 D~O ONO CDO NDO D—O
a3r+ GDH QDH GDH D~H D~H KDH b—H b—H
* * $ * * * * * * * * * * * * $ * *
* * * * * * * * *
H N N3 -: mx \0 b— (D
’OOUI UOI;€IUOOUI180&
~8JJ

16

1.0

10 * 8.0
** 1.010

05

26

0

*-

CDH

8.35
1.033 1.029
8.0

7.9
8.1

00
(DH

7".-
96*

8.65

\OO
CDH

8.45
1.032 21.032
8.0

1.030

8.2 8.1
1.035 1.035 1.035
8.0

1.008

*
7r

H
(\1

1.025 1.025

8
.020

1.020

36

(BO
(DH

* 8.15
** 1.017

L\
N

:-
N
NO
(DH

O

.1-

NO

b—H

O LIN

N

NO L00

GDH D-H

L0 L0

N LON

HO mo
GDH

O CO

N H

HO (DO

(DH COH

** **

* *

O F-

m m

1.0

7.7

1.029

1.028

1.025

 

uotietnooutisog

 

86

 

 

 

 

om.©H om.0H mN
om.NH oo.mH om.MH om
om.mH om.mH oo.NH oo.HN om.HH o:
oo.mH om.mH om.mH Nm
om.wH oo.mH Nm
oo.mH oo.NH om
om.mH om.mH om.MH om.mH oo.NH oo.NH NN
om.mH oo.NH om.MH om.mH oo.wH oo.:H HN mm
oo.wH om.NH om.:H oo.HH oo.oH oo.mH oo.mH om.HH NH n
oo.mH om.wH oo.:H oo.mH oo.:H om.mH om.NH om.:H om.mH :H m
oo.mH oo.ON 00.0 oo.:H om.NH om.wH oo.w om.wH oo.mH mH o
oo.mH oo.HN oo.oH oo.:H oo.:H om.wH oo.NH om.HN om.NH NH m
oo.mH om.NH oo.HH om.NH om.mH oo.mH om.NH om.mH om.mH HH m
oo.mH om.NH om.NH om.HH om.NH oo.HN om.NH om.mH om.mH oH 1
om.MH om.mH oo.mH oo.mH oo.:H oo.mN om.m oo.mH om.mH m m
om.mH oo.mH oo.mH oo.:H oo.:H oo.mN oo.qH oo.oH oo.UH w u
om.NH om.:H om.:H om.mH om.:H oo.HN om.:H om.mH om.HH N
oo.mH om.mH oo.©H oo.NH oo.©H om.:H oo.mH oo.MH om.mH oo.HH m
om.mH om.mH om.NH om.mH oo.uH om.NH om.mH om.mH oo.mH oo.oH m
oo.ON oo.mH oo.mH oo.mH oo.mH om.NH oo.mH om.mH oo.:H oo.m H
om.wH om.NH oo.HN oo.mH oo.mH om.mH om.wH om.NH oo.NH oo.HH m
oo.mH oo.:H om.:H oo.m oo.UH om.mH oo.NH oo.NH om.NN oo.:H N
om.mH om.MH oo.:H oo.NH oo.mH om.NH oo.mH om.NH om.HN om.:H H
_LLd
oo.mH oo.mH oo.mH om.mH mN.NH om.mH oo.mH oo.NH oo.mH om.oH mm
NNm ONN HNm NHN mHm MNN mHm mNm :Nm mHm mNNo
HthCOO HocHuQOO HochGOO

 

cOOHm .He ooH\.z.z.m.Nz cam mNmQESZ mm>Hmo

 

mm>Hmo HNpGoEHthxm
No A.z.p.mv :mNopon NmNp eoon No WHm>mH momma coHpocsm HNqu--.NH xHoszN<

87

 

 

 

 

m:.m ON.: mN
Nm.m mH.: Hm
NH.: om
NN.N m:.: mw.: mN.m Hm.m 0:
NH.: Nm.H mm.HH om
mom. NN.N mm.: NN .d
mN.m NN.N HN m
NN.N NN.N mm.m om.m Nm.w ww.: 0N m
wN.: mN.m NN.m NH u
Nm.N mm.m mm.N ON.m om.N :H.: :H w
3.: SN eNa MH m.
RN 2.: :NN OH 2
mN.: mm.m Nw.m m m
Nod m:.m N m
mN.: Nm.m mN.: N
:H.: mN.m ::.m :H.: o
mN.: mN.m mm.m mm.m m
0N.: HN.m mm.: m
NH.m ::.m NH.m N
No.: No.m NN.: :m.m mm.: m:.m mo.m H
“10
mN.m m:.m mm: 8.: NN.N. mHN NH.m Nm: Nos :3... mm
NNN QNN HNN NHN mHm mNm me mNm :Nm mHm mNmo
HOCHPCOO HOLPCOO HOQNQOO

 

woodmaz CH mosam> NEHB Namm USN meQEsz mm>Hmo

 

moCmLNoHo chHNSQHSNoEopm
pom mwsHN> mEHB mHNm mcHzocm Home COHpocsm po>HH mo moHSmmmuu.MH XHszmm<

HUN 2 2 ’0!

 

 

 

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